The present invention relates to solid support materials for the immobilization of biologically active substances thereto. In particular, the present invention relates to crosslinked polyacrylamide derivatized with functional groups to which biologically active substances can be coupled.
The immobilization of biologically active substances onto solid support materials has become an important research and diagnostic tool in various areas of biotechnology. For example, synthesis and use of solid support materials, particularly crosslinked polymer supports, having chemical structures which are physiochemically compatible with the backbone structure of a peptide has been described for use in solid-phase peptide synthesis where techniques for coupling peptides to a polymer [Stahl, et al., J. Amer. Chem. Soc., Vol. 101(18) p. 5383(1979)] and the crosslinking of various polymers [Varadarajan, et al., Biopolymers, Vol. 22, p. 839(1983)] using reverse-phase suspension polymerization in aqueous organic mixtures have been employed to obtain favorable swelling properties of such support materials in order to provide increased external and internal reaction sites bearing appropriate functional groups.
Various analytical test systems have also been described which employ solid support materials for the separation of bound and free forms of a labeled reagent in order to determine the amount of analyte present in a liquid test sample. For example, U.S. Pat. No. 4,200,436 discloses an immunoassay for the detection of antigen employing an immobilized form of the antigen to be measured which is prepared by chemically binding or physically adsorbing the antigen to solid supports or carrier materials, such as polysaccharides or plastics, according to methods known in the art. Similarly, U.S. Pat. No. 4,551,426 discloses a heterogeneous immunoassay employing an immobilized form of ouabain which is prepared by coupling ouabain, either directly or through a spacer arm such as a protein, polyamino acid, or synthetic linker, to a support material, such as beaded agarose, beaded dextran, polyacrylamide, or glass, according to methods known in the art.
Although various solid support materials are known in the art as heretofore described, polyacrylamide supports, such as crosslinked polyacrylamide gels, are particularly useful for the coupling of ligands or other biologically active substances thereto and possess a number of advantages such as low nonspecific adsorption of biological macromolecules, high chemical and thermal stability, and freedom from attack by enzymes. The structural network of crosslinked polyacrylamide gels consists mainly of segments of linear polyethylene with alternate backbone carbon atoms bearing primary amide groups which contribute to the hydrophilic character of polyacrylamide and to its low adsorption to macromolecules. Although linear polyacrylamide is water-soluble, insoluble gel networks are formed by including a bifunctional monomer in the polymerization reaction to produce crosslinkages.
In order to effectively couple a biologically active substance to such crosslinked polyacrylamide supports, it is necessary to introduce functional groups to provide reactive coupling sites. For example, crosslinked polyacrylamide [S. Hjerten and R. Mosbach, Anal. Chem. Vol. 3, p. 109(1962)] can be aminoethyl-derivatized [J. K. Inman and H. M. Dintzis, Biochemistry, Vol. 8, p. 4074(1969)] to introduce amine functional groups, or can be aminoethyldithio- or sulfhydryl-derivatized [J. K. Inman, Methods in Enzymology, Vol. 34B, p. 30(1974)] to introduce sulfhydryl functional groups.
However, such derivatized polyacrylamide support materials nevertheless contain residual amino and/or carboxylic acid groups which can result in undesirable interaction with ionic compounds and produce a gel with high buffering capacity. Such amine functional groups can also interact with amine-sensitive ligands to inactivate or decrease the binding capacity thereof to its corresponding binding partner. In particular, where the ligand coupled to an aminoethyl-derivatized polyacrylamide support is, for example, an amine-sensitive ligand such as a glycosylated peptide sequence, the ability of such ligand to bind to a corresponding antibody is substantially decreased.
Accordingly, it is an object of the present invention to provide a polyacrylamide support material derivatized with functional groups which do not nonspecifically interact with amine-sensitive ligands.
Further, it is an object of the present invention to provide a polyacrylamide support material having no residual amine or carboxyl functional groups which would otherwise interact with ionic compounds.
Another object of the present invention is to provide a polyacrylamide support material having a low buffering capacity.